Diphthamide is a post-translationally modified histidine residue found in translation elongation factor 2 (eEF-2) in all eukaryotic cells. This single modification concentrates a lot of interesting chemistry and biology. This modification involves a unique C-C bond formation reaction that links the C2 of the imidazole ring and the 3-amino-3-carboxyl propyl group derived from S-adenosylmethionine (SAM). This C-C bond formation reaction is followed by a trimethylation step and an amidation step to give the final diphthamide modification. During certain bacterial infections, this modified residue (but not the unmodified histidine residue) is specifically recognized by bacterial toxins, including diphtheria toxin and Pseudomonas exotoxin A. These toxins catalyze the transfer of an adenosine diphosphate ribose (ADP-ribose) from nicotinamide adenine dinucleotide (NAD) to one of the nitrogen atoms in the imidazole ring. The ADP-ribosylation by bacterial toxins inhibits the function of eEF-2 and thus protein synthesis, leading to cell death. Based on genetic studies, the first step of the biosynthesis, the formation of the C-C bond, requires four proteins in eukaryotes, Dph1, Dph2, Dph3, and Dph4. However, how these four proteins catalyze the reaction and why so many proteins are involved in one reaction is unknown. Heterozygous deletions of dph1 and dph4 genes are associated with several human cancers, particularly ovarian and breast cancers, raising interesting questions about the biological function of diphthamide and the tumor suppression mechanism of Dph1 and Dph4. In this proposal, we aim to study how Dph1, Dph2, Dph3, and Dph4 catalyze the first step of diphthamide biosynthesis. Understanding the biosynthesis will uncover some very interesting enzymology, as predicted by our preliminary results, and provide insights into how the biosynthesis is regulated. Understanding its biosynthesis and regulation will help to understand the biological function of this modification and the tumor suppression mechanism of Dph1 and Dph4, and may ultimately lead to the development of new ways to treat or prevent cancer. PUBLIC HEALTH RELEVANCE: Diphthamide is a unique post-translational modification that occurs in all eukaryotes. The biosynthesis of diphthamide requires multiple proteins, and mutations in several of them have been connected to cancer. This proposal aims to study the mechanism of diphthamide biosynthesis and the function of each protein required for the biosynthesis. Understanding the biosynthesis will provide important insight on the function of diphthamide, the regulation of diphthamide biosynthesis, and the mechanism of tumor formation in the absence of diphthamide biosynthesis, possibly leading to new ways to treat or prevent cancer.